Human umbilical cord blood (UCB) contains mesenchymal stem cells (MSCs) that have higher multipotentiality than adult marrow-derived MSCs (BM-MSCs). However, these cells have been difficult to obtain because the number of MSCs in UCB is extremely low (~5 to 30 out of 1 x 108 mononuclear cells). To date, the isolation of MSCs has depended upon their plastic-adhesion capacity. Most extremely immature MSCs in UCB are likely missed because their ability to adhere to plastic is poor. Our previous studies demonstrated that cellular extracellular matrix (ECM) made by bone marrow cells enhanced MSC attachment and proliferation, and retained their stem cell properties (Chen, et al, 2007, JBMR, 22:1943). Using this system, we found that human UCB may contain a large number of MSCs that adhere to the ECM (at least 10,000- to 100,000-fold greater than that previously reported), but not to plastic. More importantly, implantation of UCB-derived MSCs (UCB-MSCs) obtained by ECM adhesion into immunocompromised mice generated 3 embryonic germ layers-derived tissues including bone, muscle, fat, gland, intestine and nerve fibers. Encouraged by these findings, we propose the hypothesis that human UCB contains a large number of embryonic-like stem cells that have the potential to be used for tissue regeneration in general and myocardial reconstruction in particular. To test this hypothesis, the following 3 Specific Aims will be pursued: Specific Aim 1 is to determine the similarities and differences in global gene expression among human embryonic stem cells (hES cells), UCB-MSCs isolated by ECM adhesion versus those isolated by plastic adhesion, and human adult BM-MSCs, using microarray technology. Specific Aim 2 is to determine the ability of human UCB-MSCs obtained by ECM adhesion to selectively differentiate into desired cell lineages originated from 3 embryonic germ layers in vitro under conditions known to induce commitment to a specific cell lineage, including osteoblasts, adipocytes, chondroblasts and cardiomyocytes (mesoderm), nerve (ectoderm) and hepatocytes (endoderm). Specific Aim 3 is to evaluate the capability of transplanted UCB-MSCs obtained by the ECM adhesion to improve heart function after myocardial infarction (MI) using a well-established mouse model. Despite the great developmental potential of hES cells, there appears to be widespread agreement that a less critical source of cellular material would be preferable for the clinical use of tissue regeneration. If the proposed studies confirm that ECM can enhance retrieval of large numbers of embryonic-like stem cells from UCB, the resulting unlimited source of highly functional UCB-derived MSCs would make it feasible to be alternative to hES cells for cell-based clinical application. Specially, the ultimate outcome of these studies may be a highly practical stem cell- based therapy to treat the post-MI veteran. PUBLIC HEALTH RELEVANCE: As the US population is aging, so too is the US veteran population. According to the 2003 National Survey of Veteran Enrollees' Health and Reliance on VA, 47% of veterans were 65 years or older. Myocardial infarction (MI), as an age-related disease, is one of the leading causes of heart failure and death in the US. Currently, the best option for completely restoring cardiac function after a large MI is heart transplantation. However, it is limited by donor availability and transplant rejection. Recently, regeneration of infracted myocardium by injecting stem cells has been proposed as an alternative therapy. If the proposed studies confirm that umbilical cord blood contains a large number of embryonic- like stem cells that can be isolated by ECM adhesion procedures, and that can selectively differentiate into cardiomyocytes in vitro and reconstruct myocardium in vivo after MI, it will facilitate development of novel therapeutic approaches for cell-based regeneration to treat post-MI veteran.